Zootaxa 4276 (4): 479–502 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2017 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4276.4.2 http://zoobank.org/urn:lsid:zoobank.org:pub:E75ABE24-5B70-42E0-A240-29187F0D4140 A new of C.L. Koch, 1837 (Araneae: ) from () with the description of the male of A. pavesii Pesarini, 1991

FRANCESCO BALLARIN1,2,3,5 & PAOLO PANTINI4 1Institute of Zoology, Chinese Academy of Sciences (IOZCAS), 1 Beichen West Road, Chaoyang District, Beijing 100101, P. R. . E-mail: [email protected] 2University of Chinese Academy of Sciences, Beijing, 100049, China 3Sezione di Zoologia, Museo Civico di Storia Naturale di Verona, Lungadige Porta Vittoria, 9, I-37129 Verona, Italy 4Museo Civico di Scienze Naturali "E. Caffi" di Bergamo. Piazza Cittadella, 10, I-24129 Bergamo, Italy. E-mail: [email protected] 5Corresponding author

Abstract

A new species of the Amaurobius C.L. Koch, 1837, Amaurobius pesarinii sp. n., from the Apennine Mountains (Italy) is described on the basis of both sexes. The male of A. pavesii Pesarini, 1991, previously unknown, is also de- scribed. Females of A. pavesii and both sexes of the similar A. scopolii Thorell, 1871 are re-described. Detailed description of morphological characters allow clear separation between these species and the closely related A. ferox (Walckenaer, 1820). Their phylogenetic relationships among the most common Italian Amaurobius species are discussed on the basis of molecular data (partial fragments of the genes COI, 16S and H3), together with their distribution across the Italian Pen- insula. This is the first contribution to a multi-locus phylogenetic tree of European Amaurobius species. The synonymy of A. sciakyi Pesarini, 1991 with A. ruffoi Thaler, 1990 is proposed based on morphological characters.

Key words: Arachnida, , phylogenetic analysis, DNA

Introduction

Amaurobius C.L. Koch, 1837, is the nominal genus of the family Amaurobiidae. It contains 64 mostly Holarctic known species (World Catalog 2016). The European fauna includes 36 species (Nentwig et al. 2016), 11 of which occur in Italy: A. crassipalpis Canestrini & Pavesi, 1870, A. erberi (Keyserling, 1863), A. fenestralis (Ström, 1768), A. ferox (Walckenaer, 1830), A. jugorum L. Koch, 1868, A. obustus L. Koch, 1868, A. pallidus L. Koch, 1868, A. pavesii Pesarini, 1991, A. ruffoi Thaler, 1990, A. scopolii Thorell, 1871, and A. similis (Blackwall, 1861) (Pantini & Isaia 2016). So far, few articles have been published on the Italian amaurobiid fauna. The majority of these species remain poorly studied, and their ecology and distribution on the Italian peninsula are little understood. Furthermore, the morphological similarities between females of closely related species, and the lack of reliable diagnostic characters, have likely led to possible misidentifications, further confounding the taxonomy of the group. In his publication on the Amaurobiidae of northern Italy, Pesarini (1991) analyzed the morphology of seven Italian Amaurobius species, including A. scopolii. He also described two new : A. sciakyi from the Brescia and the Venetian Prealps, and A. pavesii (based only on female specimens) from the Apuan . The morphology and distribution of A. sciakyi closely resemble those of A. ruffoi, and the two species are treated as conspecific by Italian arachnologists; however, their synonymy has never been formalized (Trotta 2005, Pantini & Isaia 2016). A. pavesii and A. scopolii are morphologically similar, although they can be distinguished by the different shape of the epigyne (Pesarini 1991). The male of A. pavesii has not yet been described. While examining Amaurobius material from Italy, we compared numerous specimens of A. ruffoi with the type material of A. sciakyi, confirming their synonymisation based on morphology and distribution. Furthermore, we

Accepted by P. Jaeger: 10 Apr. 2017; published: 14 Jun. 2017 479 found several specimens of both sexes belonging to A. pavesii and A. scopolii. A careful examination of the samples led to the identification of the undescribed male of A. pavesii. Our observations also revealed new, detailed morphological characters in the palp, epigyne and vulva which enabled a clear separation of the two species and the closely related A. ferox. Furthermore, the material showed the presence of a new, closely related species distributed in the central Apennine mountain chain. Presumably, this new species had previously been confused with A. scopolii. This paper aims to describe the newly discovered species, and the undescribed male of A. pavesii providing detailed information on their identification. Few molecular studies have involved species of the genus Amaurobius, evident in the scarcity of sequences of amaurobiids available from public online datasets such as GenBank (GenBank 2016). So far, no specific studies on Amaurobius phylogenetics have been conducted using molecular analyses, the relationships between the European species remain unresolved. In order to genetically confirm the newly discovered species, and to shed light on its phylogenetic position, a molecular phylogenetic analysis was conducted. Our phylogeny was based on mitochondrial and nuclear gene fragments involving the most common Italian Amaurobius species. This is the first contribution to a multi-locus phylogenetic tree addressing relationships among European Amaurobius.

Material and methods

Taxonomy. A Wild M5A stereomicroscope with an ocular micrometer was used for examining and measuring specimens at the Museo Civico di Storia Naturale di Verona, Italy. Photographs of Amaurobius pesarinii sp. n. and A. scopolii were made using an Olympus Camedia E-520 digital camera mounted on an Olympus SZX16 stereomicroscope at the Zoological Museum, University of Turku, Finland. Pictures of A. ferox and A. pavesii were taken with an Olympus c7070 wide zoom digital camera mounted on an Olympus BX51 compound microscope at the Institute of Zoology, Chinese Academy of Sciences, Beijing, China. Images were combined using CombineZP image stacking software and subsequently processed with the program Photoshop CS6. To adequately see the vulva, epigynes were dissected with a needle and macerated in 10% KOH. All the specimens used for morphological analysis are preserved in 70% denatured alcohol and stored in the collections of the following institutions: MLSF=Museo di Storia Naturale, sezione di Zoologia "La Specola", Firenze, MSNB=Museo Civico di Scienze Naturali "E. Caffi" di Bergamo, MSNM=Museo Civico di Storia Naturale di Milano, MSNV=Museo Civico di Storia Naturale di Verona, ULSR=Dipartimento di Biologia Animale e dell'Uomo, Università degli Studi di Roma "La Sapienza", Italy and ZMUC=Zoological Museum, University of Copenhagen, Denmark. Leg measurements are shown as: total length (femur, patella, tibia, metatarsus, tarsus). Leg spination is reported from proximal to distal position, the possible variation in number of spines present in some of the specimens is provided in parentheses. All measurements in the text are given in millimeters. The following abbreviations are used: AM=anterior median eyes; Cd=copulatory ducts; Co=conductor; d=dorsal spines; Da=dorsal tibial apophysis; Em=embolus; Fd=fertilization duct; Ib=epigynal median plate internal bump; Im=interior margins of the epigynal median plate; Ll=lateral lobe; Ma=median apophysis; Mp=epigynal median plate; p=prolateral spines; PM=posterior median eyes; r=retrolateral spines; RTA=retrolateral tibial apophysis; Sp=spermatheca; Ta=tegular apophysis; Te=tegulum; v=ventral spines; Ve=ventral edge of the median plate. Molecular analysis and related sample collection and storage. Representative samples of the most common Italian Amaurobius species (9 species) were collected across the Italian Peninsula, with the exception of A. pallidus and one specimen of A. fenestralis, which were collected in Romania. Additional sequences were obtained from GenBank and BOLD online databases (see Appendix 1 for the detailed localities and accession codes). Live specimens were collected by hand in the field, immediately put in 95% pure ethanol, and subsequently stored at - 20°C in the Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IZCAS) after the morphological identification of the species. Total genomic DNA was extracted from a single leg of an adult specimens using a TIANamp Genomic DNA Kit (TIANGEN) following the standard manufacturer’s protocol. Amaurobius has been traditionally considered closely related to , and recent molecular studies appear to confirm this affinity (Miller et al. 2010). Two species of Callobius, C. claustrarius (Hahn, 1833) and C. sp. were therefore established as outgroups to root the tree using a sample from Romania (C. claustrarius) and sequences downloaded from GenBank (C. sp., accessions: FJ607559, FJ607450, FJ607598).

480 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI PCR amplification and sequencing. Partial fragments of the mitochondrial genes cytochrome c oxidase subunit I (COI) and 16S rRNA (16S) and the nuclear gene Histone 3 (H3) were selectively amplified using the primers and the protocols as reported in Appendix 2. The PCR amplifications were performed with an Eppendorf thermal cycler (Hamburg, ) using a final volume of 25µl. Purified PCR products were sequenced in both directions on an ABI 377 automatic sequencer (Applied Biosystems, Foster City, CA, USA) with the BigDye terminator cycle sequencing ready reaction kit. Alignment, genetic distances and phylogenetic analysis. Raw sequences were edited and assembled using BioEdit v.7.2.5 (Hall 1999). Alignment was performed on the online version of MAFFT v.7.0 (Katoh & Standley 2013) under the algorithms G-INS-i for COI and H3 and Q-INS-i for 16S and manually inspected to avoid possible mismatching. Protein-coding genes (COI, H3) were translated to proteins using MEGA v.6.0 (Tamura et al. 2013) to further check the correct alignment and the presence of potential pseudogenes. The final sequences were merged using Mesquite v.3.04 (Maddison & Maddison 2015) and the resulting combined data matrix was used to perform a Maximum likelihood (ML) analysis on the program RAxML v.8.2.0 (Stamatakis 2014). One thousand replicates of rapid bootstrap were carried out twice to select the best likelihood tree under a partition scheme by gene. A GTRGAMMA nucleotide substitution model was identified as the most appropriate model and used for each partition. The uncorrected pairwise distance between the species was calculated using MEGA v.6.0. All the sequences used in this study are deposited in GenBank (see Appendix 1 for the related codes).

Discussion

Taxonomy

Family Amaurobiidae Thorell, 1870

Genus Amaurobius C.L. Koch, 1837

Amaurobius pesarinii Ballarin & Pantini sp. n. Figs 1–3, 10, 14, 18–20, 30, 31, 35

A. scopolii Caporiacco 1930: 126 (♂ misidentification) A. scopolii Dresco 1949: 1 (♀ misidentification) A. scopolii Hubert 1965: 790, figs 7, 14, 20 (♀ misidentification) A. scopolii Dresco & Hubert 1969: 169 (♀ misidentification)

Type material. Holotype ♂: ITALY: Lazio: Roma, Canale Monterano, Regional Natural Reserve of Monterano, Guallo, 179 m, 42°08'09"N, 12°04'37"E, submediterranean oak wood, pitfall trap, 18.IX.–16.X.2007, Rizzo & Vigna Taglianti leg. (MSNV). Paratypes: ITALY: Marche: Macerata, Visso, Monte La Bandita, 1350 m, 42°53'38"N, 13°6'34"E, pitfall trap, 1♀, VI.1991–VI.1992, Pantini & Valle leg., 13♂♂, VI.–IX.1992, Pantini & Valle leg. (MSNB). Umbria: Perugia, Castiglione, Isola Polvese, Macerone, 300 m, 43°07'03"N, 12°08'19"E, 1♀, 18.V.2013, Salerno leg. (MSNB); Gualdo Tadino, San Guido, 630 m, 43°14'28"N, 12°46'58"E, 1♀, 8.XI.2015, Salerno leg. (MSNB); Terni, Piediluco, Parco di Villalago, 400 m, 42°32'08"N, 12°45'25"E, 2♀♀, 18.X.2015, Salerno leg. (MSNB). Lazio: Roma, Canale Monterano, Regional Natural Reserve of Monterano, Guallo, 179 m, submediterranean oak wood, pitfall trap, 3♀♀, 16.III.–18.IV.2007, 1♀, 18.IV.–15.V.2007, 4♂♂, 18.IX.– 16.X.2007, 1♀, 16.X.–16.XI.2007, 1♂, 1♀, 16.XI.–14.XII.2007, Rizzo & Vigna Taglianti leg. (MSNV, MSNB). Abruzzo: Teramo, Tossicia, Tozzanella, road to Colle Pelato, Gran Sasso Mountain, 1050 m, 42°31'44"N, 13°37'01"E, spruce wood, pitfall trap, 1♀, 27.VIII.2002, Marotta & Di Marco leg., 8♂♂, 1♀, 3.X.2002, Marotta & Carissimi leg., 1♀, 26.X.2002 Marotta & Matin leg. (MSNB). Other material examined. ITALY: Emilia Romagna: Bologna, Lizzano in Belvedere, Valle Dardagna, Farné, 700 m, 44°10'51"N, 10°50'39"E, 1♀, 27.XII.2013, Pedroni leg. (MSNB). Toscana: Firenze, Museo La Specola, 1♀, (as A. scopolii) 17.III.1970 (MLSF). Marche: Ascoli Piceno, Montemonaco, Isola San Biagio, 790– 1000 m, 42°54'27"N, 13°18'25"E, pitfall trap, 22♂♂, 27.VII.–1.IX.2004, 3♂♂, 1.IX.–7.X.2004, Rismondo &

NEW AMAUROBIUS SPECIES FROM ITALY Zootaxa 4276 (4) © 2017 Magnolia Press · 481 FIGURES 1–9. Male palp in ventral and retrolateral view, and male habitus, of A. pesarinii sp. n. from Canale Monterano (1– 3), A. pavesii from Bedonia (4–6), and A. scopolii from Savona (7–9).

482 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI FIGURES 10–13. Palpal tibia in disto-retrolateral view of A. pesarinii sp. n. from Canale Monterano (10), A. pavesii from Bedonia (11), A. ferox from Canale Monterano (12) and A. scopolii from Savona (13). To show diagnostic features of the Da, an orthogonal view was chosen.

Fabbri leg. (MSNB); Macerata, Bolognola, Bolognola Valley, 2♀♀, (as A. ferox), 1♀, (as A. obustus) 26.VI.1955, Biancheri leg. (MSNV); Castelsantangelo sul Nera, Gualdo, 900 m, 42°53'15"N, 13°09'35"E, pitfall trap, 13♂♂, 2♀♀, VI.–XII.1991, Buttarelli, Ghilardi, Pantini & Valle leg., 1♂, VI.–IX.1992, 3♂♂, IX.1992–VI.1993, Pantini & Valle leg. (MSNB); Castelsantangelo sul Nera, north of Rapegna village, 850 m, 42°52'07"N, 13°09'02"E, pitfall trap, 4♂♂, VI.–XII.1991, Buttarelli, Ghilardi, Pantini & Valle leg., 2♂♂, VI.–IX.1992, Pantini & Valle leg. (MSNB); same locality, 1200 m, 42°50'54"N, 13°09'15"E, pitfall trap, 2♂♂, VI.–XII.1991, Buttarelli, Ghilardi, Pantini & Valle leg., 3♂♂, VI.–IX.1992, 1♂, IX.1992–VI.1993, Pantini & Valle leg. (MSNB); Fiuminata, road to Passo Cornello, 600 m, 43°8'26"N, 12°50'54"E, pitfall trap, 7♂♂, 2♀♀, VI.–XII.1991, Buttarelli, Ghilardi, Pantini & Valle leg., 1♀, I.–VI.1992, 2♂♂, VI.–IX.1992, 1♀, IX.1992–VI.1993, Pantini & Valle leg. (MSNB); Fiuminata, Passo Cornello, 800 m, 43°7'31"N, 12°51'31"E, pitfall trap, 12♂♂, VI.–XII.1991, Buttarelli, Ghilardi, Pantini & Valle leg., 3♂♂, VI.–IX.1992, 2♂♂, IX.1992–VI.1993, Pantini & Valle leg. (MSNB); same locality, 1♀, 2.X.1992, Di Franco & Valle leg. (MSNB). Umbria: Perugia, Foligno, 42°57'20"N, 12°42'00"E, 1♀, 7.VI.1976, Bianchi leg.

NEW AMAUROBIUS SPECIES FROM ITALY Zootaxa 4276 (4) © 2017 Magnolia Press · 483 (MSNB); Gualdo Tadino, La Rocchetta, Fonte Nuova, 550 m, 43°13'43"N, 12°47'57"E, 1♀, 27.V.2013, Salerno leg. (MSNB); Gualdo Tadino, Monte Maggio, Fosso della Bastìa, 1140 m, 43°15'57"N, 12°48'15"E, 1♀, 23.IV.2014, Salerno leg. (MSNB); Gualdo Tadino, Monte Maggio, L'Impero, Buca della Neve 641 U/PG, 1175 m, 43°16'21"N, 12°47'42"E, 1♀, 11.IV.2014, Salerno & Loreti leg. (MSNB); Gualdo Tadino, , Cava del Ferro, 1070 m, 43°12'50"N, 12°48'41"E, 1♀, 24.IV.2014, Salerno & Loreti leg. (MSNB); Gualdo Tadino, Monte Serrasanta, Pian de' Cupi cave 651 U/PG, 1085 m, 43°14'17"N, 12°48'25"E, 2♀♀, 17.IV.2014, Salerno & Loreti leg. (MSNB); Gualdo Tadino, Monte Serrasanta, Pian de' Cupi, 1070 m, 43°14'17"N, 12°48'25"E, 4♀♀, 6.V.2014, Salerno leg. (MSNB); Gualdo Tadino, Valle del Fonno, Coda del Diavolo, 783 m, 43°13'35"N, 12°48'26"E, 4♀♀, 8.IV.2014, Salerno leg. (MSNB); Gualdo Tadino, Valle del Fonno, Pian della Croce, grotta dell'Ornello cave 473U/ PG, 785 m, 43°13'39"N, 12°48'44"E, 3♀♀, 20.IX.2013, 1♀, 8.IV.2014, Salerno leg. (MSNB); Nocera Umbra, Colle Aprico, Monte Pennino slope, 700 m, 43°6'53"N, 12°51'41"E, pitfall trap, 22♂♂, 2♀♀, VI.–XII.1991, Buttarelli, Ghilardi, Pantini & Valle leg., 1♀, I.–VI.1992, 5♂♂, 1♀, VI.–IX.1992, Pantini & Valle leg. (MSNB); same locality, 6♀♀, 14.VI.1992, Buttarelli, Pantini & Valle leg. (MSNB); Nocera Umbra, Colle della Croce, 1400 m, 43°6'31"N, 12°52'57"E, 8♀♀, 14.VI.1992, Buttarelli, Pantini & Valle leg. (MSNB); S. Giustino, Parnacciano, 700 m, 43°33'58"N, 12°17'34"E, 1♀, 8.VI.1991, Buttarelli, Ghilardi, Pantini & Valle leg. (MSNB); Sigillo, Monte Cucco, 1400 m, 43°21'45"N, 12°44'37"E, 9♀♀, 15.VI.1992, Buttarelli, Pantini & Valle leg. (MSNB); Sigillo, Piani di Monte, 1200 m, 43°21'29"N, 12°45'00"E, pitfall trap, 1♂, VI–XII.1991, Buttarelli, Ghilardi, Pantini & Valle leg., 1♂, IX.1992–VI.1993, Pantini & Valle leg. (MSNB); Sigillo, north of the village, 550 m, 43°20'21"N, 12°44'58"E, pitfall trap, 4♂♂, 1♀, VI.–XII.1991, Buttarelli, Ghilardi, Pantini & Valle leg., 2♂♂, VI.–IX.1992, Pantini & Valle leg. (MSNB); Terni, Lugnano in Teverina, Sibilla Cave, 1♂, 13.XI.1929, Andreini leg. (MLSF) (Caporiacco 1930: as A. scopolii). Lazio: Frosinone, Guarcino, Vermiciano Mountain, Gnomo Cave, 100 m, 1♂, 23.VIII.2003, Pedicone leg. (MSNB); Roma, Canale Monterano, Regional Natural Reserve of Monterano, Diosilla, 255 m, 42°07'53"N, 12°05'25"E, deciduous forest with Black Alder and hornbeam, pitfall trap, 1♂, 18.IX.– 16.X.2007, 1♀, 14.XII.2007–15.I.2008, Rizzo & Vigna leg. (ULSR); Canale Monterano, Regional Natural Reserve of Monterano, Mola Ceccarelli, 250 m, 42°8'N, 12°3'E, beechwood, pitfall trap, 1♂, 16.X.–16.XI.2007, 1♀, 14.XII.2007–15.I.2008, Rizzo & Vigna leg. (ULSR); Cervara di Roma, 41°59'22"N, 13°04'07"E, 1♀, 29.VII.2009, La Casella leg. (MSNB). Abruzzo: L’Aquila, near Pietrasecca cave, 750 m, 42°08'13"N, 13°07'42"E, 1♂, 26.V.1991, Bologna leg. (MSNB); Teramo, Castelli, between Colle Rustico and Fondo della Salsa, 900–1050 m, 42°27'38"N, 13°42'54"E, 4♀♀, 24.VII.2004, Magnati & Pantini leg. (MSNB); Isola del Gran Sasso d'Italia, Gran Sasso Mountain, road to Pagliara lake, 900 m, 42°28'22"N, 13°41'37"E, mixed wood, 1♂, 3.X.2002, Marotta & Carissimi leg., 1♂, 28.VIII.2003, 2♂♂, 1♀, 7.X.2003, Marotta leg. (MSNB); Rocca Santa Maria, road to Valle Castellana, Monti della Laga, 655–850 m, 42°42'13"N, 13°29'59"E, mixed wood, 1♀, 25.VII.2001, 1♂, 1♀, 28.X.2001, 1♂, 6.X.2002, Marotta leg. (MSNB). Etymology. The specific epithet is a patronym in honor of the Italian arachnologist Carlo Pesarini who extensively contributed to the study of the Italian spider fauna, including the family Amaurobiidae. Diagnosis. Amaurobius pesarinii sp. n. is closely related to A. pavesii, A. scopolii and A. ferox, but can be distinguished from them by its lighter coloration. It also has a smaller body size in comparison with A. scopolii and A. ferox. Males of A. pesarinii sp. n. can be recognized by the unique shape of Da, being more trapezoidal and without a clear indentation, unlike in males of A. scopolii and A. pavesii, and lacking of a dorsal hook as present in A. ferox (Fig. 10 vs. Figs 11–13). In addition, Ta is reduced and only faintly protruding from the tegulum in A. pesarinii sp. n. being more prominent in other species (Fig. 14 vs. Figs 15–17). Females of A. pesarinii sp. n. can be distinguished by the shape of Mp, being narrower and less U-shaped than Mp of A. scopolii (Figs 18, 31 vs. Figs 24, 33) and less V-shaped and lacking a clear squared tip as Mp of A. pavesii (Figs 18, 31 vs. Figs 21, 32). Moreover, Ll is pointed in A. pesarinii sp. n. and rounded in A. scopolii (Figs 18, 31 vs. Figs 21, 32). When observed posteriorly, Mp of A. pesarinii sp. n. shows a straight ventral edge, lacking the clear concavity present in other species (Fig. 20 vs. Figs 23, 26, 29). Additional characters to separate the new species from A. scopolii are: Im always partially visible when viewing the epigyne ventrally (Figs 18, 31 vs. Figs 24, 33), and Ib with a bump in the interior side (absent in A. scopolii; Figs 19, 35 vs. Figs 25, 37). Females of A. pesarinii sp. n. can be distinguished from those of A. ferox by the shorter Ll (at least as long as the Mp in A. ferox; Figs 18, 31 vs. Figs 27, 34). Diagnostic characters of the species are summarized in Table 2. Description. Male. Holotype: Total length: 8.39. Carapace 3.67 long, 2.81 wide. Paratypes: (Based on 10 specimens from different areas) Total length: 6.75–9.33. Carapace 3.32–4.44 long, 2.4–3.0 wide.

484 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI FIGURES 14–17. Palpal bulbs in proximo-ventral view of A. pesarinii sp. nov from Sigillo (14), A. pavesii from Bedonia (15) and A. scopolii from Savona (16) and from Bobbio (17), highlighting the diagnostic shape of the Ta in the different species.

Habitus as in Fig. 3. Carapace yellowish-brown with central darker striae; eye region brown. AM smaller than PM. Chelicerae light brownish, frontally swollen, with 5 anterior and 3 posterior teeth. Labium and gnathocoxae uniformly light brown, sternum yellowish-brown as the carapace, without pattern. Dorsal side of the abdomen gray with yellowish cardiac mark and additional markings of the same color, ventral side gray with yellowish markings. Legs uniformly yellowish-brown. Leg lengths: I 12.57 (3.23, 1.27, 3.26, 3.24, 1.57), II 10.57 (3.20, 1.25, 2.44, 2.42, 1.26), III 8.90 (2.81, 1.11, 2.01, 2.02, 0.95), IV 11.32 (3.21, 1.25, 2.80, 2.81, 1.25). Leg formula: I IV II III. Leg spination as in Table 1. Palp as in Figs 1–2, 10, 14, yellowish-brown, RTA elongated, rectangular, almost as long as the tibia. Da distally oriented, massive, with a roughly trapezoidal shape, tip lightly bent onwards. Sparse serration on the distal margin, with 2–3 shallow indentations more or less visible in different specimens. Te globular. Ma approximately 2 times longer than wide, ending with a sharp tip. Conductor wide. Ta little developed with roughly triangular shape, only the anterior part slightly protruding orthogonally from the surface of the tegulum. Embolus with blunt tip. Female. (Based on 20 specimens from different areas) Total length: 6.97–9.30 Carapace 3.64–4.69 long, 2.18– 3.10 wide.

NEW AMAUROBIUS SPECIES FROM ITALY Zootaxa 4276 (4) © 2017 Magnolia Press · 485 FIGURES 18–30. Female genitalia: A. pesarinii sp. n. from Canale Monterano epigyne in ventral view (18), vulva in dorsal (19) and posterior (20) view; A. pavesii from Tornolo epigyne in ventral view (21), vulva in dorsal (22) and posterior (23) view; A. scopolii from Savona epigyne in ventral view (24), vulva in dorsal (25) and posterior (26) view; A. ferox from Canale Monterano, epigyne in ventral view (27), vulva in dorsal (28) and posterior (29) view; A. pesarinii sp. n. from Canale Monterano epigyne in ventral view showing the mating plug (red arrow) (30).

486 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI FIGURES 31–38. Comparison of female genitalia: A. pesarinii sp. n. from Castiglione del Lago epigyne in ventral view (31), from Canale Monterano vulva in dorsal view (35); A. pavesii from Bonassola epigyne in ventral view (32), from Tornolo vulva in dorsal view (33); A. scopolii from Bobbio epigyne in ventral view (36), from Savona vulva in dorsal view (37); A. ferox from Cernusco Montevecchia epigyne in ventral view (34), from Canale Monterano vulva in dorsal view (38).

NEW AMAUROBIUS SPECIES FROM ITALY Zootaxa 4276 (4) © 2017 Magnolia Press · 487 TABLE 1. Leg spination of the Amaurobius species discussed in the text. Unusual numbers of spines present in some of the specimens are in parentheses. Amaurobius pesarinii sp. n. Male Leg Femur Patella Tibia Metatarsus I d1 r1 p1 r0 p0 r1-1 p1-1 v2-2-2 r1-1 p1-1-1 v2-2-3 II d1- r1-1 p1 r0 p0 r1-1 p1-1 v2-2-2 r1-1-1 p1-1-1 v2-2-3 III d1 r1 p1 r0 p1 r-1-1 p1-1 v2(1)-2-2 r1-1-1 p1-1-1 v2-2-3 IV d1 r1 p1 p0 r0 r1 p1 v1-1-2 r1-1 p1-1-1 v2-2-3 Female Leg Femur Patella Tibia Metatarsus I d1 r0 p1 r0 p0 r1-1 p1-1 v2-2-2 r1-1 p1-1 v2(1)-2-3 II d1-(1) r1-(1) p1 r0 p0 r1-1 p1-1 v2-2-2 r1-1 p1-1 v2-2-3(2) III d1(0) r1 p1 r0 p1 r1-1 p1-1(0) v1-1-2 r1-1-1 p1-1-1 v2(1)-2-3 IV d1 r1 p0 p0 r0 r1-1 p0 v1-1-2(1) r1-1 p1-1 v2-2-2 Amaurobius pavesii Male leg Femur Patella Tibia Metatarsus I d1 r1 p1 r0 p0 r1 p1-1 v2-2-2 r1-1 p1-1 v2-2-3 II d1-1 r1 p1 r0 p0 r1-1 p1-1 v2-2-2 r1-1 p1-1 v2-2-3 III d1-1 r1 p1-1 r1 p1 r1-1 p1-1 v2-2-2 r1-1-(1) p1-1-(1) v2-2-3 IV d1 r1 p1 p0 r0 r1-1 p1-1 v2-1-2(1) r1 p1 v2(3)-2-3 Female leg Femur Patella Tibia Metatarsus I d1 r0 p1 r0 p0 r1 p1-1-(1) v2-2-2 r1-1 p1-1 v2-2-3 II d1-1 r1-1 p1 r0 p0 r1-1 p1-1 v2-2-2 r1-1 p1-1 v2-2-3 III d1-1-(1) r1 p1 r0 p0 r1-1 p1-1-(1) v1-1-2 r1-1 p1-1 v2-2-3 IV d1-1-(1) r1 p0 p0 r0 r0-1 p1-1 v1-1-2(1) r1-1 p1-1 v2-2-3 Amaurobius scopolii Male leg Femur Patella Tibia Metatarsus I d1 r1-1(0) p1-1 r0 p0 r(1)-1-1 p(1)-1-1 v2-2-2 r1-1-1 p1-1-1 v2-2-3 II d1-1 r1-1 p1-1 r0 p1 r(0)-1-1 p1-1 v2-2-2 r1-1-1 p1-1-1 v2-2-3 III d1-1-1 r1-1 p(0)-1-1 r1 p1 r1-1 p1-1 v2-2(1)-2 r1-1-1 p1-1-1 v2-2-3 IV d1-1 r1 p1 r1 p0 r1-1 p1-1 v1-2-2 r1-1 p1-1-1 v2-2-2 Female leg Femur Patella Tibia Metatarsus I d1 r1 p0 r0 p0 r1-1 p1-1 v2-2-2 r1-1 p1-1 v2-2-3 II d1-1(0) r1-1 p1-1-1 r0 p0 r1-1 p1-1 v2-2-2 r1-1 p1-1 v2-2(1)-3 III d1-1-(1)- r1-1 p1-1-1 r0 p0 r1-1 p1-1 v1-2(1)-2 r1-1-1 p1-1-1 v2-1-3 IV d1-1 r1 p0 p0 r0 r1-1 p1 v1-1-2 r0(1)-1 p2 v2(1)-1

488 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI TABLE 2. Summary of the diagnostic characters and measurements in male and female of A. pesarinii sp. n., A. pavesii, A. scopolii and A. ferox.

Amaurobius pesarinii Amaurobius Amaurobius sp. n. pavesii scopolii

7.06–7.70 / 6.83– 9.5–10.2 / 10.52– Body length 6.75–9.33 / 6.97–9.30 8–10 / 11–15 8.86 12.2

Body color yellowish-brown brown brown dark brown Male/Female

triangular and Dorsal tibial trapezoidal, the tip is triangular, massive massive, ending triangular apophysis (Da) lightly bend onwards and stumpy with a hook-like dorsal process Dorsal tibial with 2 deep with 2 deep marks, with 2 deep marks apophysis with 2–3 faint marks marks of the same the proximal of the same size serration size deeper wide, ridge- little developed, ridge-shaped with shaped, protruding wide, ridge-shaped, triangular, only the a blunt bulge, orthogonally protruding Palp Tegular distal part is lightly protruding (sometimes orthogonally from apophysis (Ta) protruding orthogonally orthogonally from diagonally) from the surface of the from the surface of the the surface of the the surface of the tegulum tegulum tegulum tegulum massive, slender, massive and wide, massive, approximately approximately 1.5 approximately 2 approximately 1.5 Median apophysis 1.5 times longer than times longer than times longer than times longer than (Ma) wide, ending with a wide, ending with wide, ending with wide, ending with a sharp tip a blunt tip a blunt tip blunt tip strongly V- approximately V- Median plate approximately V- shaped, with a large, with the shaped with a blunt (Mp) shaped, with a blunt tip clearly squared shape of a wide U tip tip Bump on the interior side of well developed well developed absent well developed median plate (Ib) Interior margin partially visible, widely well visible, only not visible, always not visible, always of the median covered by the median half-covered by covered by the covered by the plate (Im) plate the median plate median plate median plate Epigyne Ventral edge of the median plate straight concave concave concave (Ve) long and pointed, very short and short and pointed, short and pointed, as long or longer squared, shorter shorter than the Lateral lobes (Ll) shorter than the median than the median than the median median plate, plate, protruding inward plate, protruding plate protruding inward inward

Carapace yellowish-brown with central darker striae, eye region brown. AM smaller than PM. Chelicerae brown, frontally swollen, with 5 anterior and 3 posterior teeth. Labium and gnathocoxae brown. Sternum uniformly yellowish-brown as the carapace, without pattern. Abdomen dorsally gray with yellowish cardiac mark and additional markings of the same color. Ventral side uniformly gray with yellowish markings. Legs uniformly yellowish-brown. Leg lengths: I 10.15 (2.85, 1.26, 2.51, 2.28, 1.25), II 8.58 (2.52, 1.25, 2.01, 1.79, 1.01), III 8.12 (2.84, 1.11, 1.53, 1.76, 0.88), IV 9.67 (2.85, 1.25, 2.28, 2.28, 1.01). Leg formula: I IV II III. Leg spination as in Table 1. Epigyne and vulva as in Figs 18–20, 30–31, 35. Mp roughly V-shaped in ventral view and roughly X-shaped in posterior view; posterior tip blunt and rounded, Ve straight. Im partially visible. Ib well-developed. Ll weakly developed, shorter than margin of Mp, ending with blunt median tooth. Sp rounded, Fd short, Cd bent inward. Ecology. Amaurobius pesarinii sp. n. can be found in Apennine deciduous and mixed forests rich in debris, large stones and litter. The elevational range of the species seems to extend approximately from 150 m to 1500 m a.s.l., probably excluding the flatlands. It has occasionally been collected in caves. Distribution. Italy, endemic to the Apennines. Amaurobius pesarinii sp. n. is distributed throughout the

NEW AMAUROBIUS SPECIES FROM ITALY Zootaxa 4276 (4) © 2017 Magnolia Press · 489 central-southern Italian peninsula along the Apennine mountain chain, with the exception of the Ligurian Apennines and the northern parts of the Tuscan-Emilian Apennines where it is replaced by the closely related species A. scopolii and A. pavesii. No records are known for Sicily or Sardinia (Fig. 39). Additional notes. In the past, Amaurobius pesarinii sp. n. has often been confused with A. scopolii due to the general similarities between the two species. All the records of A. scopolii from central-south Italy are likely to have been A. pesarinii sp. n. The drawings reported by Hubert (1965, figs. 14 and 20) can be clearly attributed to females of A. pesarinii sp. n. judging by the shape of the median plate. Furthermore, the distribution of A. scopolii seems limited only to the south-western Alps and northern Apennines, no records of this species have been confirmed from the central-southern part of the Apennines, where instead A. pesarinii sp. n. is abundant. Further details concerning potential misidentifications can be found in the discussion of A. scopolii.

Amaurobius pavesii Pesarini, 1991 Figs 4–6, 11, 15, 21–23, 32, 36

A. pavesii Pesarini 1991: 275, figs 20a–b (♀) A. scopolii Pesarini 1991: 272, figs 14a–b (♂ misidentification)

Type material. Holotype ♂: ITALY: Toscana: Lucca, Monte Pania della Croce, 44°02'07"N, 10°19'24" E, 1 ♀, 23.VI.1983, Sciaky leg. (MSNM). Paratypes: ITALY: Emilia Romagna: Medesano, Felegara, 44°42'31.3"N, 10°05'45"E, 1♀, 19.III.1983, Pavesi leg. (MSNM). Toscana: Monte Forato, 44°00'51.1"N, 10°20'08.8"E, 1♀, 22.VI.1983, Sciaky leg. (MSNM). Other material examined. ITALY: : Genova, Borzonasca, Rio della Lora, 1000 m, 44°26'57"N, 9°27'02"E, 2♀♀, 25.V.2010, Lodovici & Pantini leg. (MSNB). Borzonasca, Passo del Bocco, 1000 m, 44°24'54"N, 9°26'38"E, 3♀♀, 15.VII.2001, Pantini leg. (MSNB); La Spezia, Bonassola, between Framura and Bonassola villages, 80 m, 44°11'21"N, 9°34'16"E, 3♀♀, 9.III.2003, Pantini leg. (MSNB); Portovenere, Palmaria island, 170 m, 44°02'36"N, 9°50'40"E, 3♀♀, 24–28.IV.2003, Ciocca leg. (MSNB); Varese Ligure, Passo Cento Croci, 1000 m, 44°25'13"N, 9°37'25"E, 2♂♂, 1♀, IX.1991–V.1992, Buttarelli, Cerbino, Pantini & Valle leg. (MSNB). Emilia Romagna: Parma, Albareto, Spallavera, 600 m, 44°27'37"N, 9°41'25"E, oak wood, 8♀♀, 2.XI.2015, Bergamaschi, Lodovici, Massaro & Pantini leg. (MNBG); Bedonia, Passo Montevacà, 800 m, 44°32'06"N, 9°36'33"E, pitfall trap, 1♂, VI.–IX.1992, 1♀, XI.1992–IV.1993, Pantini & Valle leg. (MSNB); Tornolo, Monte Zatta, 1050 m, 44°24'45"N, 9°28'40"E, beechwood, 3♀♀, 2.XI.2015, Bergamaschi, Lodovici, Massaro & Pantini leg. (MNBG); Piacenza, Carpaneto Piacentino, Badagnano, 44°50'58"N, 9°46'08"E, 1♂, IX.1983, 1♂, 15.X.1983, Pavesi leg. (MSNM) (Pesarini 1991: as A. scopolii). Toscana: Massa-Carrara, Carrara, Campo Cecina, 1350 m, 44°6'40"N, 10°6'50"E, 1♀, 14.IV.1991, Valle leg. (MSNB); Piazza al Serchio, Fosso Carpinelli, 44°12'00"N, 10°14'34"E), 3♀♀, 20.IX.1975, Valle & Moretti leg. (MSNB); Vagli di Sotto, 643 m, 44°06'58"N, 10°18'06"E, under stones in a deciduous forest, 1♀, 14.VIII.2015, Ballarin & Gaiga leg. (MSNV); Villa Collemandina, Canigiano village, 1042 m, 44°10'46"N, 10°23'21"E, under stones in a deciduous forest, 1♀, 14.VIII.2015, Ballarin & Gaiga leg. (MSNV). Diagnosis. Amaurobius pavesii is closely related to A. pesarinii sp. n., A. scopolii and A. ferox but smaller in size than the last two. The darker coloration (Fig. 6 vs. Fig. 3), the triangular Da with 2 clear indentations in the distal margin (Fig. 11 vs. Fig. 10), and the wider Ta bearing a stocky bulge (Fig. 15 vs. Fig. 14) enable the separation of males of A. pavesii from males of A. pesarinii sp. n. (Da without a clear indentation and Ta reduced, lacking a bulge, in A. pesarinii sp. n.). Males of A. pavesii can be distinguished from those of A. scopolii by the smaller size and by the shorter Ta with a blunt bulge (bulge absent in A. scopolii; Fig. 15 vs. Figs 16–17). Additionally, hook-like dorsal process in Da is missing in A. pavesii (present in A. ferox; Fig. 11 vs. Fig. 12). Females of A. pavesii can be easily separated from those of A. pesarinii sp. n., A. scopolii and A. ferox by the different appearance of Mp, shaped as narrow V with clear squared tip in A. pavesii (wider and rounded in the other species; Figs 21, 32 vs. Figs 18, 24, 27, 31, 33–34). Other diagnostic characters are: Im wide, clearly visible at the side of the Mp (poorly visible in A. pesarinii sp. n. and covered by Mp in A. scopolii and A. ferox) and Ll short and rounded (pointed in the other species; Figs 21, 32 vs. Figs 18, 24, 27, 31, 33–34). Moreover, Ve shows a pronounced concavity when Mp is observed posteriorly, which is absent in A. pesarinii sp. n. (Fig. 23 vs. Fig. 20). Diagnostic characters of the species are summarized in Table 2.

490 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI Description. Male. (Based on 3 specimens from Bedonia and Varese Ligure) Total length: 7.06–7.70 Carapace 3.63–4.01 long, 2.50–2.55 wide. Habitus as in Fig. 6. Carapace brown with central darker striae, eye region dark brown. AM smaller than PM. Chelicerae dark brown, frontally swollen, with 5 anterior and 3 posterior teeth. Labium and gnathocoxae uniformly brown, sternum lighter without pattern. Dorsal side of abdomen gray with lighter cardiac mark and additional markings of the same color, ventral side gray with lighter markings. Legs uniformly brown. Leg length: I 12.57 (4.59, 1.56, 2.50, 2.55, 1.37), II 12.32 (3.65, 1.27, 3.05, 3.07, 1.28), III 10.13 (3.20, 1.25, 2.27, 2.28, 1.13), IV 12.45 (3.61, 1.27, 3.14, 3.17, 1.26). Leg formula: I, IV, II, III. Leg spination as in Table 1. Palp as in Figs 4–5, 11, 15; brown, RTA elongated, rectangular, almost as long as tibia. Da distally oriented, massive, triangular, with 2 indentations of the same size on distal margin. Te globular. Ma massive, approximately 2 times longer than wide, with blunt tip. Conductor wide. Ta ridge-shaped with blunt bulge, protruding orthogonally from surface of tegulum. Embolus with slightly enlarged, trapezoid tip. Redescription. Female. (Based on 10 specimens from different localities) Total length: 6.83–8.86. Carapace 3.50–4.25 long, 2.03–2.72 wide. Carapace yellowish-brown with central darker striae, eye region brown. AM smaller than PM. Chelicerae dark brown, frontally swollen, with 5 anterior and 3 posterior teeth. Labium and gnathocoxae dark brown. Sternum uniformly yellowish-brown as the carapace, without pattern. Abdomen dorsally gray with yellowish cardiac mark and additional markings of the same color. Ventral side uniformly gray with yellowish markings. Legs uniformly yellowish-brown. Leg length: I 11.43 (3.44, 1.40, 2.79, 2.54, 1.26), II 9.51 (2.84, 1.26, 2.25, 2.03, 1.13), III 7.29 (2.52, 1.09, 1.76, 1.03, 0.89, 7.29), IV 10.56 (3.15, 1.26, 2.51, 2.52, 1.12). Leg formula: I IV II III. Leg spination as in Table 1. Epigyne and vulva as in Figs 21–23, 32, 36. Mp narrow, V-shaped, roughly triangular in posterior view with clearly squared tip and concave Ve. Im clearly visible when Mp observed ventrally. Ib well-developed. Ll very short and rounded, shorter than Mp. Sp rounded, Fd short. Cd stumpy, bent inward. Ecology. Likewise the other two related species, A. pavesii lives in mixed and deciduous forests where it builds webs under superficial stones, logs and debris. Its elevational range extends from the lowlands up to 1500 m. Distribution. Italy, endemic of the northern Apennines. Amaurobius pavesii is present only in a restricted area in the northern Apennines where it partially overlaps with A. scopolii (Fig. 39). Additional notes. Pesarini (1991) described the species based only on a few females collected in western Emilia Romagna and northern Toscana regions. In the description, the author underlined the peculiar shape of the epigyne, which would allow an easy separation from the related Amaurobius scopolii and A. ferox. Drawings of the epigyne and vulva of the paratype were also provided (p. 274, figs 20a–b). Our observations of the material we collected support the author’s preliminary observations: the shape of the epigyne is diagnostic and limited only to female specimens living in the area where the species was originally found. Furthermore, both morphological and molecular analysis support A. pavesii as a separate, valid species (Fig. 40) despite its limited distribution, partially overlapping with the distribution of A. scopolii. The male palp illustrated by Pesarini (1991) as A. scopolii (p. 272, figs 14a–b) actually refers to A. pavesii.

Amaurobius scopolii Thorell, 1871 Figs 7–9, 13, 16–17, 24–26, 33, 37

A. scopolii Thorell 1871: 206 (♂♀). A. scopolii Simon 1914: 36, 39, 59, figs 61–62, 76 (♂♀). A. scopolii Pesarini 1991: 275, figs 19a–b (♀; ♂= A. pavesii).

Material examined. ITALY: Lombardia: Pavia, Brallo di Pergola, near hotel Colletta, beechwood, 1380 m, 44°43'21"N, 9°15'28"E, 3♀♀, 20.VI.2001, Ferrario, Lodovici & Pantini leg., 2♀♀, 31.VII.2001, Facheris, Ferrario, Bianchi & Pantini leg. (MSNB). Piemonte: Cuneo, Aisone, Vallone della Valletta, 1400–1674 m, 44°17'06"N, 7°13'50"E, 2♂♂, 1♀, 17.VIII.2010, Isaia & Vigna Taglianti leg. (MSNB); Garessio, 44°12'31"N, 8°00'02"E, 1♂, 1♀, 1.V.2004, 1♂, 1♀, 3.X.2004, Beikes leg. (MSNB); Roccabruna, 44°29'30"N, 7°19'35"E, 1♀, 23.V.1999, Isaia leg. (MSNB); Trezzo Tinella, Bric Boscasso, 650 m, 44°38'42"N, 8°07'25"E 1♀, 17.XII.1993, Delmastro leg. (MSNM); Valdieri, Piano del Valasco, 1700 m, 44°12'10"N, 7°14'44"E, under stones in a mixed

NEW AMAUROBIUS SPECIES FROM ITALY Zootaxa 4276 (4) © 2017 Magnolia Press · 491 forest, 3♂♂, 3♀♀, 27.VIII.2014, Ballarin leg. (MSNV), 1♂, 2♀♀, 27.VIII.2014, Pantini leg. (MSNB); Valdieri, Terme di Valdieri, 44°12'25"N, 7°16'13"E, 1♀, 16.VI.1988, Sciaky leg. (MSNM); Valdieri, Vallone di Lourousa, 44°12'06"N, 7°17'28"E, 1♂, 2♀♀, 29.VII.2009, Isaia leg. (MSNB); Vernante, Sotterranei Militari di Vernante, 44°15'18"N, 7°31'48"E, 1♂, 19.IX.2007, Isaia leg. (MSNB); Vinadio, 44°18'29"N, 7°10'14"E, 1♀, 16.VI.1988, Sciaky leg. (MSNM); Val Bormida, 1♀, 23.IV.2003 Isaia leg. (MSNB). Liguria: Genova, Borzonasca, Giaiette, 850 m, 44°25'03''N, 9°28'08''E, beechwood, pitfall trap, 1♀, 31.X.2009–25.V.2010, 1♀, 25.V.–18.VIII.2010, Lodovici, Pantini & Valle leg. (MSNB); same locality 2♀♀, 25.V.2010, Lodovici & Pantini leg. (MSNB); Imperia, Triora, rio Lazzarin near Verdeggia, 1075 m, 44°02'29"N, 7°43'12"E, 1♀, 9.X.2001, Bergamo Museum Staff leg. (MSNB); Savona, Andora, Marina di Andora, 60 m, 43°57'33"N, 8° 8'45"E, wood, pitfall trap, 4♂♂, 1♀, 23.IV.2005–26.IV.2006, Fabbri leg. (MSNB); same locality, 1♀, 23.IX.2005, 3♀♀, 26.IV.2006, Fabbri leg. (MSNB); Borgio Verezzi, 44°09'39"N, 8°18'47"E, 1♀, 15.VI.1956, Valle leg. (MSNB); Bormida, country road n°15 to Colle Melogno, 670–700 m, 44°15'34"N, 8°13'19"E, 5♀♀, 17.VII.2001, 1♀, 11.X.2001, Bergamo Museum Staff leg. (MSNB); Calizzano, Colle Quazzo, 800 m, 44°13'07"N, 8°05'13"E, beechwood, 1♂, 7♀♀, 11.X.2001, Ferrario & Pantini leg. (MSNB, MSNV); Calizzano, road to Colle Quazzo, 800 m, 44°13'07"N, 8°05'13"E, 1♀, 18.VII.2001, 1♀, 11.X.2001, Bergamo Museum Staff leg. (MSNB); Calizzano, near torrente Frassino, 920 m, 44°14'06"N, 8°11'44"E, 2♀♀, 11.X.2001, Bergamo Museum Staff leg. (MSNB); Savona, Monte San Giorgio, 44°22'41"N, 8°24'58"E, 5♀♀, 12.II.1983, Pavesi leg. (MSNM); , Monte Beigua, 1200 m, 44°26'1"N, 8°33'52"E, 3♀♀, 17.VII.2001, Bergamo Museum Staff leg. (MSNB); , 44°21'59"N, 8°34'39"E, 1♀, 22.IV.1956, Malanchini leg. (MSNB); Emilia Romagna: Parma, Corniglio, Bosco di Corniglio, 44°28'15"N, 10°04'51"E, wood, 1♀, 28.VI.1949, Valle leg. (MSNB); Tornolo, Monte Zatta, 1050 m, 44°24'45"N, 9°28'40"E, beechwood, 3♀♀, 2.XI.2015, Bergamaschi, Lodovici, Massaro & Pantini leg. (MNBG); Piacenza, Bobbio, Monte Pernice, 44°47'46"N, 9°19'40"E, 1♀, 1.V.1967, Valle leg. (MSNB); Bobbio, Passo Pernice, 1100 m, 44°47'46"N, 9°19'40"E, wood, pitfall trap, 12♀♀, 20.V.–20.VI.2001, 1♂, 31.VII.–19.IX.2001, 1♀, 20.III.–26.IV.2002, 1♀, 26.IV.–27.VI.2002, Pantini leg. (MSNB); same locality, 8♀♀, 20.V.2001, Magnati & Pantini leg., 3♀, 31.VII.2001, Bianchi, Facheris, Ferrario & Pantini leg., 3♂♂, 3♀♀, 19.IX.2001, Pantini leg., 2 ♀♀, 20.III.2001, Lodovici & Pantini leg. (MSNB); Bobbio, below Passo Penice, 1050 m, 44°48'15"N, 9°20'35"E, 1♀, 1.V.1967, Valle leg., 2♀♀, 20.V.2001, Magnati & Pantini leg. (MSNB); Bobbio, along the road to Monte Pernice, 1400 m, 44°47'12"N, 9°19'08"E, pitfall trap, 5♂♂, 2♀♀, 19.IX.2001–20.III.2002, 2♀♀, 26.IV.–27.VI.2002 Pantini leg. (MSNB); same locality, 6♀♀, 20.VI.2001, Ferrario, Lodovici & Pantini leg., 4♀♀, 31.VII.2001, Bianchi, Facheris, Ferrario & Pantini leg. (MSNB); Travo, Monte Pietra Parcellara, 44°50'39"N, 9°28'24"E, 1♀, 14.XI.1982, Brenia leg. (MSNM). Other material examined. Amaurobius occidentalis Simon, 1892; : Bragança: Algosinho, 1♀; Freixo de Espada a Cinta, 1♂; Mogadouro, 1♂, 2♀♀, (no further data present) (ZMUC) (all identified as A. scopolii). Amaurobius ferox: ITALY: Lombardia: Como, Cernusco Montevecchia, 270 m, 1♀, 25.IV.1963, Bianchi leg. (MSNB). Lazio: Roma: Canale Monterano, Regional Natural Reserve of Monterano, Diosilla, 255 m, 42°07'53"N, 12°05'25"E, deciduous forest with black alder and hornbeam, pitfall trap, 1♂, 1♀, 18.IV.–15.V.2007, Rizzo & Vigna leg. (MSNV). Diagnosis. Amaurobius scopolii is closely related to A. pavesii, A. pesarinii sp. n. and A. ferox but larger in size than the first two. Males of this species can be distinguished from those of A. pesarinii sp. n. and A. pavesii by larger Ta, extending beyond ventral margin in retrolateral view being smaller and less protruding in the other two species (Figs 16–17 vs. Figs 14–15). The darker color (Fig. 9 vs. Fig. 3), the longer and slimmer Ma (Fig. 8 vs. Fig. 2) and the stumpier, triangular Da bearing 2 clear, deep indentations (without clear indentation in the other species) (Fig. 13 vs. Fig. 10) distinguish males of A. scopolii from those of A. pesarinii sp. n. Lack of hook-like dorsal process in Ta allows quick separation from A. ferox (Fig. 13 vs. Fig. 12). Females of A. scopolii can be distinguished from those of A. pesarinii sp. n., A. pavesii and A. ferox by the wider U-shaped Mp always covering Im (Figs 24, 33 vs. Figs 18, 21, 27 and 31–32, 34) and by the absence of Ib (Figs 25, 37 vs. Figs 19, 22, 28 and 35–36, 38) (Mp V-shaped and Ib well developed in the other species). Ve with clear concavity further distinguishes A. scopolii from A. pesarinii sp. n. (Ve straight in the latter species; Fig. 26 vs. Fig. 20). Ll shorter than Mp in A. scopolii is in contrast to Ll at least as long as Mp in A. ferox (Figs 24, 33 vs. Figs 27, 34). Diagnostic characters of the species are summarized in Table 2.

492 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI Redescription. Male. (Based on 10 specimens from different localities) Total length: 9.50–10.20. Carapace 4.41–5.15 long, 3.12–3.48 wide. Habitus as in Fig. 9. Carapace brown with central darker striae, eye region dark brown. AM smaller than PM. Chelicerae dark brown, swollen in front, with 5 anterior and 3 posterior teeth. Labium and gnathocoxae brownish, sternum lighter without pattern. Abdomen dorsally dark-gray with a faint yellowish cardiac mark and additional markings of the same color, ventral side yellowish-gray. Legs uniformly brown. Leg lengths: I 17.19 (4.45, 1.5, 4.5, 4.8, 1.94), II 13.14 (3.8, 1.4, 3.12, 3.28, 1.54), III 10.81 (3.22, 1.15, 2.42, 2.75, 1.27), IV 13.55 (3.8, 1.28, 3.32, 3.64, 1.51). Leg formula: I, IV, II, III. Leg spination as in Table 1. Palp as in Figs 7–8, 13, 16–17, brown. RTA elongated, rectangular, almost as long as the tibia. Da distally oriented, massive and stumpy, triangular-shaped, with 2 deep indentations in the distal margin, the proximal deeper and more visible than the distal. Te globular. Ma massive, approximately 3 times longer than wide, with a blunt tip. Conductor wide. Ta wide, ridge-shaped, protruding orthogonally from the surface of the tegulum (in a few specimens protruding more diagonally, see Fig. 17). Embolus with enlarged, trapezoid tip. Female. (Based on 10 specimens from different localities) Total length: 10.52–12.20. Carapace 4.83–5.27 long, 3.20–3.61 wide. Carapace brown with central darker striae, eye region dark brown. AM smaller than PM. Chelicerae dark brown, frontally swollen, with 5 anterior and 3 posterior teeth. Labium and gnathocoxae dark brown. Sternum uniformly yellowish-brown, without pattern. Abdomen dorsally gray with yellow cardiac mark and additional markings of the same color, ventral side yellowish-brown. Legs uniformly yellowish-brown. Leg length: I 13.42 (3.76, 1.59, 3.28, 3.18, 1.61), II 11.29 (3.36, 1.51, 2.55, 2.51, 1.37), III 9.72 (3.02, 1.34, 2.07, 2.15, 1.16), IV 12.38 (3.8, 1.43, 2.9, 2.93, 1.32). Leg formula: I, IV, II, III. Leg spination as in Table 1. Epigyne and vulva as in Figs 24–26, 33, 37. Mp large, U-shaped, roughly rectangular in posterior view, Ve clearly concave. Im not visible in any view. Ib absent. Sl weakly developed, shorter than the margins of Mp, ending with a blunt tooth protruding inward. Sp rounded, Fd short. Cd slightly bent inward. Ecology. Amaurobius scopolii is associated with many types of habitats, from deciduous forests to open areas, with a preference for places rich in stones and debris. It can also occur occasionally at the entrance of caves (Brignoli 1972). Its elevational range extends from the lowlands approximately up to 1700 m. Distribution. Italy and , endemic to the south-western Alps/northern Apennines. Amaurobius scopolii is distributed from the French Maritime Alps to the northern part of the Tuscan-Emilian Apennines, where its range partially overlaps with A. pavesii (Fig. 39). Records from other European countries are very likely misidentifications of other local Amaurobius species, records from central-south Italy refer to the similar A. pesarinii sp. n. Additional notes. Amaurobius scopolii was originally described by Thorell (1871) from southeastern France. Its presence in French Maritime Alps is confirmed by later authors (Simon 1914; Le Peru 2007). The species was correctly recognized by Simon (1914). In his work on French spiders he properly reports the diagnostic serration present in the distal margin of the tibial dorsal apophysis of the male of this species ["bordée d'une carène noire à bord tranchant crénelée d'échancrures inégalement espacées, sur sa face antérieure"], as further illustrated in his drawing (p. 35, fig. 62). In Italy, A. scopolii has been reported from different localities in the entire peninsula, especially in caves, as summarized by Brignoli (1972). However, most of these records are likely to be misidentifications of similar species, including A. pesarinii sp. n. For example, Caporiacco (1930: 126) incorrectly assigns a male of A. pesarinii sp. n. from Umbria to A. scopolii. Hubert (1965: 791, 794, figs 14, 20) reports 2 females of A. scopolii from south Italy (Potenza, Caserta) and one for the north (Novara) and illustrates 2 epigynes without specifying the locality of the drawn samples. The original material collected by Hubert (1965) is probably lost and therefore cannot be used to comment the author’s findings. However, the illustrated epigynes show a wide V-shaped median plate, which identify them as belonging to A. pesarinii sp. n. All the other known records of A. scopolii from central to south Italy (Dresco 1949, Dresco & Hubert 1969) are based only on females or juveniles, which can be easily misidentified without a deeper analysis of the diagnostic characters. No other recent specimens of A. scopolii have been found in south Italy. All old records are therefore likely to be misidentifications of A. pesarinii sp. n. Material reported by Caporiacco (1940) for the Veneto region includes five specimens (1 male and 4 females) of A. scopolii. The original samples have been lost during WWII (Ruffo S., personal communication), and so far no more specimens of this species have been found in the Veneto region despite extensive searching (Ballarin et al.

NEW AMAUROBIUS SPECIES FROM ITALY Zootaxa 4276 (4) © 2017 Magnolia Press · 493 2011). Therefore these records can be considered misinterpretations of Caporiacco. Two specimens collected by Caporiacco in the Friuli Venezia Giulia region, preserved in La Specola museum, are juveniles and therefore cannot be identified to species level. Few specimens are reported from the Lepontine Alps and Brescia Prealps (Isaia et al. 2007), although no other records are known from the surrounding area including Aosta Valley or . The presence of this species in the north-western Alps is therefore doubtful. Different senior authors reported A. scopolii for several European countries such as Iberian Peninsula (Bacelar 1927, Barrientos & Ferrandez 1982), (Kratochvíl 1935), (Nikolic & Polenec 1981), , and some north Aegean Greek islands (Strand 1916: as Amaurobius ?scopolii). These records are often based on old data. We examined material from Portugal (males and females) identified as A. scopolii and they are in fact A. occidentalis. Although it is not possible for us to confirm all the other records from the remaining European countries, it is likely that they are misidentifications of other Amaurobius species.

FIGURE 39. Distribution of A. pesarinii sp. n. (red dots and squares), A. scopolii (white dots) and A. pavesii (blue dots) in Italy. Red squares refer to unconfirmed A. pesarinii sp. n. localities as reported by Dresco (1949), Hubert (1965) and Dresco & Huber (1969).

494 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI Amaurobius ruffoi Thaler, 1990

A. ruffoi Thaler 1990: 242, figs 4–6, 9–13 (♂♀). A. sciakyi Pesarini 1991: 267, figs 9a–b, 10a–b (♂♀ misidentification) syn. n. A. ruffoi Thaler & Knoflach 1998: 38, fig. 17 (♂).

Material examined. ITALY: Lombardia: Brescia, Pezzoro, 1♂, 23.IV.1988, Sciaky leg. (holotype A. sciakyi, MSNM). Pizzocolo mountain, 1♀, 7.V.1983, Pavesi leg. (paratype A. sciakyi, MSNM). Veneto: Verona, Giazza, Grotta dei Prusti Cave, 1♀, 25.III.1982, Sciaky leg. (paratype A. sciakyi, MSNM). Sant’Anna d’Alfaedo, Vajo della Marchiora, 450 m, 1♂, 25.XI.2009, Sette leg. (MSNV). Spluga della Preta Cave, 2♂♂, 3♀♀, 19.V.1990; 2♀♀, 18.II.1989; 2♀♀, 24.II.1990, Zanon leg. (paratypes A. sciakyi, MSNM). Velo veronese, Varalta, 900 m, under stones in deciduous forest, 1♂, 1♀, 7.XI.2007, Ballarin leg. (MSNV). Vicenza, Monte Summano, Sasso della Madonna, beechwood forest litter, 1105 m, 1♀, 16.III.1998, Fontana leg. (MSNB). Schio, Tretto, Vermech, 470 m, 1♂, 9.V.1998, Fontana leg. (MSNB). Additional notes. In 1990 Thaler described a new species of Amaurobius for Italy, A. ruffoi Thaler, 1990 from Grappa and Lessini mountains in Veneto region. The following year Pesarini (1991) described another species, A. sciakyi Pesarini, 1991, from the same area and the close Brescia Prealps. The two species appear morphologically very similar, and Italian arachnologists have speculated that they are probably in synonymy (Caoduro et al. 1994: 33, Trotta 2005: 75, Ballarin et al. 2011: 18, Pantini & Isaia 2016). While examining material used in the current study, we also examined the holotype and paratypes of A. sciakyi preserved in the Museo Civico di Storia Naturale di Milano, and compared them with A. ruffoi specimens from the type locality and surrounding areas. A careful examination of the morphological characters demonstrates that there are no significant differences between A. sciakyi and A. ruffoi. Based on this evidence, as well as the overlapping distribution of the species, there is no doubt that A. sciakyi Pesarini, 1991 and A. ruffoi Thaler, 1990 are conspecific; therefore their synonymy is proposed.

Phylogenetic analysis

A total of 12 Amaurobius species were included in the phylogenetic analysis. Taxon sampling included all currently recognized Italian Amaurobius species, with the exception of A. obustus of which no fresh samples or online sequences were available. The presence of A. similis in the Italian peninsula is uncertain, being limited only to old records (Pesarini 1991), nevertheless we decided to include it in the analysis to evaluate its relationship with the other Italian species. A final dataset of 1986 base pairs (COI: 1227bp, 16S: 434bp, H3: 325bp) was analyzed; the phylogenetic tree obtained from the maximum likelihood (ML) analysis is shown in Fig. 40. The uncorrected pairwise genetic distances between species is shown in Appendix 3. The Italian Amaurobius species cluster into two main clades (bootstrap support value 100%) corresponding to two different evolutionary lineages, the “ ferox- group” and the “fenestralis-group”, the first consisting of A. ferox, A. pavesii, A. pesarinii sp. n., A. scopolii, A. jugorum and A. crassipalpis and the second of A. fenestralis, A. similis, A. ruffoi, A. erberi and A. pallidus. The species belonging to the first clade are grouped in two further lineages with high support value (100%) suggesting a closer relationship between A. jugorum and A. crassipalpis. The assumption of A. pavesii, A. pesarinii sp. n., A. scopolii, and A. ferox as distinct, valid species with a common ancestor is strongly supported by molecular evidence in addition to the morphology (see Fig. 40 and appendix 3). Moreover, the molecular analysis reveals the presence of a further possible new species (Amaurobius sp.) from the northern Apennines closely related to A. jugorum but clearly separated from it. The ferox-group gathers the wide majority of the endemic Amaurobius species (A. crassipalpis, A. pavesii, A. pesarinii sp. n., A. scopolii, Amaurobius sp.), most of which are distributed in different areas of the Apennines and Alps, with limited or without overlapping distributions. We speculate that the particular structure and orientation of the mountain chains within the Italian peninsula, as well as relatively recent climatic events (e.g. the Pleistocene glaciations), may have influenced the evolutionary history of this group. Nevertheless, to test this hypothesis a more comprehensive study including all European species, a wider pool of gene fragments and a precise time calibration of the phylogenetic tree, are necessary. The second major clade, including the representatives of the ”fenestralis group”, has a general low bootstrap support in the internal nodes, preventing reliable assumptions from being made about the evolutionary relationships within this clade. The second clade mostly includes species with a wider distribution in , which suggests a possible different phylogeographic history compared to the ferox-group.

NEW AMAUROBIUS SPECIES FROM ITALY Zootaxa 4276 (4) © 2017 Magnolia Press · 495 The presence of two distinct species groups within the Italian Amarobius species was already assumed by Pesarini (1991: 262–263) and partially by Thaler (1990), using morphological differences present in the palpal tibial apophysis, palpal tegular apophysis and epigynal median plate. Our phylogeny does not reject these hypotheses. Based on our results and Pesarini’s original descriptions, we re-define the diagnostic characters of the groups as the following: ferox-group: palp with only one dorsal tibial apophysis, thick and more or less trapezoidal with a serrated distal margin and, in some species, an elongated dorsal hook-like protuberance; tegular apophysis ridge-shaped and simple; epigynal median plate approximately as long as wide, with a pointed or distinctly rounded posterior margin; fenestralis-group: palp with two dorsal tibial apophyses, the centro-dorsal one shorter, blunt or pointed, the latero-dorsal longer and always pointed; tegular apophysis wide, not ridge-shaped and divided into two parts; epigynal plate wider than long, with a flat or lightly rounded posterior margin.

FIGURE 40. Phylogenetic tree of the most common Italian Amaurobius species showing the two major species-groups discussed in the text: ferox-group (red) and fenestralis-group (blue). The newly described species is reported in bold. The code before the name of each species refers to the specimen locality reported in Appendix 1. Branch lengths are scaled in relation to the number of substitutions per site, numbers at nodes denote bootstrap support according to Maximum Likelihood.

Conclusions

Because of their general morphological similarity, Amaurobius scopolii, A. pavesii and A. pesarinii sp. n. have been often confused by senior arachnologists, particularly when descriptions have been based on females only. Males and females of all these species have different levels of intraspecific variation, especially in the shape of the

496 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI palpal tegular apophysis and epigynal median plate, which probably has been an additional cause of misidentification. Nevertheless, a careful morphological analysis reveals several diagnostic characters which allow a clear separation of the species. According to our studies, the real distribution of A. scopolii seems to be limited to the south-western Alps (including the French Maritime Alps) and the northern Apennines where it overlaps with A. pavesii. Old data from other European countries should be considered as probably mistaken. A. pesarinii sp. n., on the contrary, is distributed all along the remaining Apennine chain, and no clear evidence of overlapping distribution with the other two species is known. Our molecular phylogeny supports a close relationship between A. scopolii, A. pavesii, A. pesarinii sp. n. and A. ferox, and their possible common origin. It also makes the phylogenetic position of Italian Amaurobius species more definite, supporting their separation into two main clades. A further potential species, recognized on the base of molecular evidence, remains undescribed for now since the only available specimen (a female) does not allow a correct morphological identification. The presence of possible new species highlights how little is known about the genus Amaurobius in Italy and its diversity, which could be wider than previously expected. Additional collections on the Italian peninsula, and deeper molecular and morphological studies including other European species, can probably shed further light on this genus and its evolutionary history.

Acknowledgments

The authors are particularly thankful to Shuqiang Li, Seppo Koponen and Leonardo Latella for allowing the use of microscopes and other facilities at the Institute of Zoology of the Chinese Academy of Sciences, the Zoological museum of the University of Turku, and the Museo Civico di Storia Naturale di Verona respectively. Many thanks to Yuri Marusik for his kind suggestions and precious advice on the species morphology, and Vikki Smith for kindly checking the English text of an early version of the manuscript. We express our warm gratitude to Nikolaj Scharff (Zoological Museum, University of Copenhagen), Monica Leonardi (Museo Civico di Storia Naturale di Milano), Luca Bartolozzi and Sarah Whitman (Museo di Storia Naturale di Firenze, sezione di Zoologia "La Specola"), Augusto Vigna Taglianti and Camilla De Stefanis (Dipartimento di Biologia Animale e dell'Uomo, Università degli Studi di Roma "La Sapienza") and Marco Isaia (Dipartimento di Biologia Animale e dell'Uomo, Università degli Studi di Torino) for allowing access to the material stored in their collections. We are sincerely thankful to Elena Pelizzoli for the illustrations, and equally grateful to all the people who kindly helped during the field work in Italy and Romania or directly collected specimens for the molecular analysis, in particular: Rodolfo Ballarin, Bruna Bergamaschi, Manuele Gaiga, Omar Lodovici, Melania Massaro, Augustin Nae, Piero Salerno and Zhe Zhao. The manuscript benefitted greatly from comments by Peter Jäger, Marco Isaia, and two anonymous reviewers.

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500 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI APPENDIX 2. Primers and PCR protocols used in this study. Gene Primers Or. Sequence (5'–3') Reference PCR protocol fragment COI LCO1490 F GGTCAACAAATCATCATAA Folmer et al. 1994 94°(3'); 94°(30''), 45°(30''), AGATATTGG 72°(45'')x5; 94°(3'); 94°(40''), CHR2 R GGATGGCCAAAAAATCAA Barrett & Herbert 50°(40''), 72°(1')x35; 72°(5') AATAAATG 2005 C1-J-2183 F CAACATTTATTTTGATTTTT Simon et al. 1994 TGG C1-N-2776 R GGATAATCAGAATATCGTC Hedin & Maddison GAGG 2001 16S 16SA F CGCCTGTTTATCAAAAACA Arnedo et al., 2001 94°(1'); 94°(40''), 45°(40''), T 72°(1')x35; 72°(5') 16SB2 R CTCCGGTTTGAACTCAGAT Arnedo et al. 2001 CA H3 H3aF F ATGGCTCGTACCAAGCAGA Colgan et al. 1998 94°(5'); 94°(40''), 50°(40''), CVGC 72°(1')x35; 72°(5') H3aR R ATATCCTTRGGCATRATRGT Colgan et al. 1998 GAC

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502 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI